Immunometric assays using monoclonal antibodies

ABSTRACT

&#34;Two-site&#34; or &#34;sandwich&#34; immunometric assay techniques for determination of the presence and/or concentration of antigenic substances in fluids using monoclonal antibodies are described and compared to conventional assays using polyclonal antibodies. Also described are inhibition assays using complexes of antigens with a monoclonal antibody.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of co-pending applicationSer. No. 175,133, filed Aug. 4, 1980, now U.S. Pat. No. 4,376,110, thedisclosure of which is incorporated by reference.

FIELD OF THE INVENTION

This invention relates to methods for detecting and/or determining theconcentration of antigenic substances in fluids such as serum. Inanother aspect it relates to immunometric and inhibition assaytechniques. In yet another aspect it relates to monoclonal antibodies.

BACKGROUND

The determination of the presence or concentration of antigenicsubstances, for example, those associated with a wide variety ofphysiological disorders, in serum or other body fluids reliesincreasingly upon immunoassay techniques. These techniques are basedupon formation of a complex between the antigenic substance beingassayed and an antibody or antibodies in which one or the other memberof the complex may be labelled, for example, by a radioactive elementsuch as ¹²⁵ I, which permits its detection and/or quantitative analysisafter separation of the complexed labelled antigen or antibody fromuncomplexed labelled antigen or antibody.

In the case of a competition immunoassay technique, the antigenicsubstance in a sample of fluid being tested for its presence competeswith a known quantity of labelled antigen for a limited quantity ofantibody binding sites. Thus, the amount of labelled antigen bound tothe antibody is inversely proportional to the amount of antigen in thesample. By contrast, immunometric assays employ a labelled antibody. Insuch an assay, the amount of labelled antibody associated with thecomplex is proportional to the amount of antigenic substance in thefluid sample.

Immunometric assays have been found to be particularly well suited forthe detection of polyvalent antigens, i.e., antigenic substances thatare able to complex with two or more antibodies at the same time. Suchassays typically employ a quantity of unlabelled antibody bound to asolid support that is insoluble in the fluid being tested and a quantityof soluble antibody bearing a label such as a radioactive isotope thatpermits detection and/or a quantitative estimate of the amount of theternary complex formed between solid phase antibody, antigen, andlabelled antibody.

In immunometric assays known to the prior art, typically "forward"assays, in which the antibody bound to the solid phase is firstcontacted with the sample being tested to extract the antigen from thesample by formation of a binary solid phase antibody: antigen complex,are employed. After a suitable incubation period, the solid support iswashed to remove the residue of the fluid sample, including unreactedantigen, if any, and then contacted with a solution containing a knownquantity of labelled antibody.

After a second incubation period to permit the labelled antibody tocomplex with the antigen bound to the solid support through theunlabelled antibody, the solid support is washed a second time to removethe unreacted labelled antibody. In a simple "yes/no" assay to determinewhether the antigen is present in the sample being tested, the washedsolid support is tested to detect the presence of labelled antibody, forexample, by measuring emitted radiation if the label is a radioactiveelement. The amount of labelled antibody detected is compared to thatfor a negative control sample known to be free of the antigen. Detectionof labelled antibody in amounts substantially above the backgroundlevels indicated by the negative control is interpreted to indicate thepresence of the suspect antigen. Quantitative determinations can be madeby comparing the measure of labelled antibody with that obtained forcalibrated samples containing known quantities of the antigen.

This kind of assay is frequently referred to as a "two-site" or"sandwich" assay since the antigen has two antibodies bonded to itssurface at different locations. This and related techniques aredescribed by Wide at pp. 199-206 of "Radioimmunoassay Methods," Editedby Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970. An assaybased on this technique for the detection of the antigen associated withserum hepatitis using an ¹²⁵ I labelled antibody is described in U.S.Pat. No. 3,867,517.

Despite their great utility, the prior art immunometric assays have beenrecognized to be slow procedures, in part because two washing steps arerequired and because lengthy incubation periods are required to approachequilibrium, i.e., the point at which the amount of complex formed doesnot change with increasing time.

To eliminate at least one of the washing steps associated with thisprocedure, so-called "simultaneous" and "reverse" assays have beenproposed. A simultaneous assay involves a single incubation step as theantibody bound to the solid support and the labelled antibody are bothadded to the sample being tested at the same time. After the incubationis completed, the solid support is washed to remove the residue of fluidsample and uncomplexed labelled antibody. The presence of labelledantibody associated with the solid support is then determined as itwould be in a conventional "forward" sandwich assay.

A reverse assay involves the stepwise addition first of a solution oflabelled antibody to the fluid sample followed by the addition ofunlabelled antibody bound to a solid support after a suitable incubationperiod. After a second incubation, the solid phase is washed inconventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labelled antibody. Thedetermination of labelled antibody associated with the solid support isthen determined as in the simultaneous and forward assays.

Both the simultaneous and reverse assay techniques require a sufficientexcess amount of solid phase antibody to bind most or all of the antigenpresent to avoid a high dose hook effect where artificially negative orlow quantitation of antigen is observed at extremely high concentrationof antigen. For this reason, the forward assay has been the approachpreferred by the prior art. That is because large amounts of highlypurified, active antibody specific to the antigen of interest forpreparing a solid phase with sufficient antigen binding capacity isdifficult to obtain from the "polyclonal" antibodies used in prior artprocesses. When an immunogenic substance is introduced into a livingbody, the body's immune system reacts by generating antibodies to everysite on the immunogen it recognizes. A large immunogenic proteinmolecule may have dozens of sites and a foreign cell may have hundreds.Thus, while each antibody producing cell produces antibody specific fora single antigenic site the immune system has generated a specie ofspecific antibody producing cells for each immunogenic site recognized.In addition, the body has produced relatively large quantities ofantibodies to antigens other than the one of interest such that most ofthe antibody in the polyclonal mixture is not specific for the antigenof interest. Accordingly, the antibodies used in prior immunometricassays are necessarily "polyclonal" in nature since the antibodies arederived from antisera raised in a conventional manner in animals andtheir purification is difficult. Methods for affinity purifying suchantibodies have generally been time consuming and resulted in low yieldsand loss of high affinity antibodies.

When employing conventional polyclonal antibody mixtures in the reverseand simultaneous assays, the formation of a "sandwich" comprising theantigen complexed by two or more labelled antibodies which complex withthe antigen at different sites is possible. These complexes could remainsoluble in the sample being tested, be removed by subsequent washingsteps, and not "counted" when the solid phase is analyzed for solidphase bound labelled antibody. If this happens to a significant extent,sensitivity of the assay is reduced and erroneous results may arise.However, if the unlabelled bound antibody is added to the sample firstas in the forward sandwich assay, steric considerations preventformation of a sandwich comprising the antigen complexed to two or moreunlabelled antibodies where labelled antibody is excluded from alsobinding to the antigen. Accordingly, the antigen is free to react with alabelled antibody molecule. Nevertheless, it has been proposed to use asimultaneous assay for human thyroid stimulating hormone (HTSH) byemploying a large excess of the unlabelled antibody bound to a solidphase to minimize formation of a soluble complex by soluble labelledantibodies. See Jeong et al., "Comparison of Radioimmunoassay (RIA) witha Unique, Single-Incubation Two-Site Immunoradiometric Assay (IRMA) asApplied to the Determination of Human Thyroid Stimulating Hormone(HTSH)," Bio-Rad Laboratories, 1979.

A variation of a simultaneous assay is described in U.S. Pat. No.4,174,384. In that assay, separate portions of Anti-IgG (Human) arelabelled, respectively, with a fluorescing chromophore (fluorescein) anda chromophore (rhodamine) which absorbs light emitted by thefluorescein. Both antibodies, in a soluble form, are contacted with asample containing human IgG. Reaction of the Anti-IgG with the IgG maybring the two chromophores close enough together, i.e., within 100angstroms or less, that the emission of light by the fluorescingchromophore is absorbed (quenched) by the other. The percentage ofmaximum fluorescence for the sample is determined and used as a measureof the amount of IgG in the sample.

It has also been proposed to use a reverse assay for HTSH, hepatitisassociated antigen (HAA) and carcinoembryonic antigen (CEA) by employinga quantity of labelled antibody sufficient to assure a labelledantibody: antigen complex but insufficient to form a "sandwich" of allthe antigen present in a sample. See U.S. Pat. No. 4,098,876.

Since all three of the procedures known to the prior art use apolyclonal mixture of antibodies, the potential for cross-reaction withother materials in serum or other fluid than the antigen for which thetest is intended is increased. The occurrence of cross-reactivity withother antigens also reduces the sensitivity of the test for the suspectantigen and increases the prospect of a "false-positive" assay.Furthermore, the use of polyclonal antibodies in a simultaneous orreverse assay requires a careful consideration of the amount of labelledantibody used relative to the amount of solid phase antibody and/orantigen present. In the case of using fluorescence quenching,sensitivity is reduced because the minimum spacing between thefluorescing chromophore and the quenching chromophore is not assuredwhen polyclonal antibodies are employed.

In view of these shortcomings, the limitations to the immunometricprocedures known to the prior art are readily apparent. The conventionalforward assay is time consuming; the simultaneous and reverse assays areaccomplished with fewer steps but require large quantities of solidphase specific antibody and are not well suited to determination ofsmall concentrations of antigen since formation of a sandwich of theantigen with a multiple number of labelled antibody molecules competeswith formation of the sandwich comprising boundantibody:antigen:labelled antibody or, in the case of using fluorescencequenching, the formation of a sandwich without pairing of a fluorescentchromophore with a quenching chromophore is possible; and all aresubject to misinterpretation of false-positives due to the polyclonalnature of the antibody.

Accordingly, one object of the present invention is to provide animproved process for the immunometric assay for antigenic substances.

More specifically, an object of the present invention is to provide morerapid immunometric assay techniques.

Another object of the present invention is to provide more sensitiveimmunometric assay techniques.

Yet another object of the present invention is to provide improved"simultaneous" and "reverse" immunometric assays.

A further object of the invention is to provide improved inhibitionassays.

The manner in which these and other objects are realized by the presentinvention will be apparent from the summary and detailed description setforth below.

SUMMARY OF THE INVENTION

According to the present invention, the polyclonal antibodies used in animmunometric assay, for example, as the unlabelled antibody bound to asolid support and the antibody used as the soluble labelled antibody or,in the case of assays relying upon fluorescence quenching, theantibodies carrying a fluorescing or quenching chromophore are replacedby at least one and usually two or more different monoclonal antibodies,i.e., each antibody specific to a single antigenic site and separatelyproduced by clones derived from unique cell lines. In a preferredembodiment of the invention, the monoclonal antibody used as theantibody bound to a solid support is the product of a different cellline than is the monoclonal antibody used for the labelled antibody andthe two monoclonal antibodies are selected to bind the antigenicsubstance at sites remote from each other so as to not interfere withthe other's binding to the antigen. In the case of fluorescencequenching, the two antibodies are also usually the products of differentcell lines and are selected so as to not interfere with the other'sbinding yet bring the two chromophores close enough together to permitquenching of fluorescence, i.e., usually to within about 100 angstroms.The advantages of the present invention, particularly in simultaneousand reverse assays, over prior art methods will become clear afterconsideration of the accompanying drawings and the following detaileddescription of the invention.

Also, according to the present invention, monoclonal antibodies areemployed in inhibition assays. In such assays, a known quantity of anantigen and monoclonal antibody is contacted with a sample suspected ofcontaining an antigen corresponding to the known antigen added with themonoclonal antibody. The extent to which inhibition of the complexbetween the antibody and antigen occurs because a complex comprising themonoclonal antibody and antigen from the sample is formed is a measureof the presence and/or amount of antigen in the sample assayed. In apreferred embodiment, the antibody and antigen are bound, respectively,to one of the members of a pair of fluorescing and quenchingchromophores. Inhibition of the formation of a complex between thelabelled antigen and antibody by antigens in the sample being assayedleads to a reduction in quenching and an increase in fluorescence. Theextent of the inhibition of quenching is a measure of antigenconcentration in the sample. In another preferred embodiment of aninhibition assay, the known antigen and antibody the original complexare bound to particles, for example, latex particles, of a size whichpermits agglomerates to form. When a sample suspected of containingantigen is contacted with the antibody and bound antigen, inhibition ofagglomerate formation occurs because of complexing between the boundantibody and sample antigen which cannot form agglomerates. Thereduction in agglomeration can be measured using turbidimetrictechniques.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the results obtained using polyclonalantibodies in four types of immunometric assay for human IgE.

FIG. 2 is a similar graph illustrating the difference in resultsobtained using monoclonal antibodies in the same four types ofimmunometric assay for human IgE.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, according to the present invention, the polyclonalantibody used in an immunometric assay for an antigenic substance isreplaced by a monoclonal antibody. Similarly, monoclonal antibodies areused in inhibition assays. The present invention is useful for thedetermination of the presence and concentration of a wide variety ofantigenic substances including polyvalent antigenic substances.Accordingly, as used herein, the term antigen or antigenic substancerefers broadly to substances to which antibodies can be produced. Amongsuch substances may be mentioned haptens, hormones such as insulin andhuman thyroid stimulating hormone (HTSH), gamma globulins, allergens,viruses, virus subunits, bacteria, toxins such as those associated withtetanus and with animal venoms, and even some drugs. Among the specificantigens which may be assayed by the processes of the present inventionmay be mentioned carcinoembryonic antigen (CEA), hepatitis A and B,hepatitis Non A-Non B, IgE and alphafetoprotein.

The monoclonal antibodies useful in the present invention are obtainedby the process discussed by Milstein and Kohler and reported in Nature256 495-497, 1975. The details of this process are well known and willnot be repeated here. However, basically it involves injecting a mouse,or other suitable animal, with an immunogen. The mouse is subsequentlysacrificed and cells taken from its spleen are fused with myeloma cells.The results is a hybrid cell, referred to as a "hybridoma," thatreproduces in vitro. The population of hybridomas is screened andmanipulated so as to isolate individual clones each of which secretes asingle antibody species to the antigen. Each individual antibody speciesobtained in this way is the product of a single B cell from the immuneanimal generated in response to a specific antigenic site recognized onthe immunogenic substance.

When an immunogenic substance is introduced into a living host, thehost's immune system responds by producing antibodies to all therecognizable sites on the substance. This "shotgun" approach toproducing antibodies to combat the invader results in the production ofantibodies of differing affinities and specificities for the immunogenicsubstance. Accordingly, after the different hybridoma cell lines arescreened to identify those that produce antibody to the desired antigen,the antibodies produced by the individual hybridoma cell lines arepreferably screened to identify those having the highest affinity forthe immunogenic substance stimulating their original production beforeselection for use in the present invention. Selection based on thiscriterion is believed to help provide the increased sensitivity in theimmunometric and inhibition assays of the present invention usingmonoclonal antibody compared to the polyclonal antibody used in theprior art which, at best, has an affinity for the antigen which isroughly the average of the affinities of all antibodies produced by theimmune system. Preferably, the monoclonal antibody selected will have anaffinity compatible with the desired sensitivity and range for the testsystem under consideration. Preferably the antibody will have anaffinity of at least 10⁸ liters/mole and, more preferably, an affinityof at least about 10⁹ liters/mole.

Furthermore, those monoclonal antibodies having the highest affinitiescan be further screened by running a simulated assay on specimens knownto give false positive-results with processes employing conventionalpolyclonal antibodies to identify those monoclonal antibodies which donot cross-react and give false positive results.

Because the two-site immunometric assay relies upon formation of anantibody:antigen:antibody sandwich, usually two different monoclonalantibodies which do not interfere with the binding of each other to theantigen are selected to be the bound antibody and the soluble labelledantibody or the antibody pair when fluorescence quenching is used. Sinceboth are necessary to complete the sandwich, reverse and simultaneousassays can be conducted without concern, for example, that a complex oflabelled antibody:antigen:labelled antibody will form which willpreclude formation of a complex between the antigen and the antibodybound to the solid phase and therein lies a particular advantage of thepresent invention. Furthermore, a forward assay can be accomplishedwithout the intermediate washing step since the two antibodies bind totwo different sites. We refer to such a process as a "fast forward"assay.

However, particularly in the case of a forward assay, the samemonoclonal antibody can be used for both the labelled antibody and theantibody bound to the solid support when the antigenic substancepossesses identical antibody binding sites sufficiently remote from eachother to allow more than one antibody molecule to be bound at the sametime. In such a system, the addition first of the bound antibody to thesample precludes formation of a sandwich because of stericconsiderations. When the labelled monoclonal antibody is subsequentlyadded, it is also able to complex with the antigen bound to unlabelledantibody on the solid phase.

The unlabelled monoclonal antibody used in the process of the presentinvention to extract the antigenic substance from the sample beingtested may be immobilized on any of the common supports used inimmunometric assays. Among these may be mentioned filter paper, plasticbeads or test tubes made from polyethylene, polystyrene, polypropyleneor other suitable material. Also useful are particulate materials suchas agarose, cross-linked dextran, and other polysaccharides. Thetechniques for bonding antibodies to such materials are well know tothose skilled in the art. For example, antibodies may be bound topolysaccharide polymers using the process described in U.S. Pat. No.3,645,852.

The labelled monoclonal antibody used in the present invention may beprovided with the same labels used in prior art immunometric assays.Among these may be mentiond fluorogenic labels for detection byfluorimetry as described in U.S. Pat. No. 3,940,475 and enzymaticmarkers as described in U.S. Pat. No. 3,654,090. It is presentlypreferred to label the antibody with a radioisotope such as ¹²⁵ I using,for example, the procedure of Hunter and Greenwood, Nature, 144 (1962),page 945 or that of David et al., Biochemistry, Vol. 13, pp. 1014-1021,1974.

In a typical assay, the amount of labelled antibody associated with theinsoluble sandwich complex is determined by examination of the insolublecarrier material by suitable means. However, it is also possible torelate the presence or absence of antigen in the fluid sample beingassayed to the amount of labelled antibody which does not react duringthe assay and remains in soluble form.

The advantages of the present invention in which monoclonal antibodiesare used in immunometric assays as compared to polyclonal antibodies areseen by reference to the following example. In this example, fourcomparative assays, a simultaneous assay, a reverse assay, a forwardassay, and a "fast" forward assay, were run using both monoclonalantibody and polyclonal antibody using a standard serum containing 100IU/ml of human IgE as the positive sample. Normal horse serum containingno IgE was used as a negative control.

The polyclonal antibody to IgE used as the labelled antibody in theexample was obtained from Pharmacia Diagnostics of Piscataway, N.J. Thepolyclonal antibody bound to the solid support was obtained from Tago,Inc. of Burlingame, Calif.

Monoclonal antibody to IgE was obtained using the method of Milstein andKohler discussed above. The two antibodies selected each exhibited anaffinity for IgE of greater than 10⁹ liters/mole and did not interferewith the other's binding to IgE.

The assays were run using unlabelled antibody bound to agarose by theprocess of U.S. Pat. No. 3,645,852. Labelling of antibody was by ¹²⁵ Iaccording to the process of David et al. referred to above. Phosphatebuffered saline, pH 7.4, was used to wash all samples.

EXAMPLE

(1) Simultaneous Assay Method

Duplicate samples were run in which 100 μl of a suspension of antibodyimmobilized on agarose particles was mixed with 100 μl of specimen(serum) and 100 μl of soluble ¹²⁵ I-labelled antibody. This mixture wasincubated for the specified times shown in Table I (for polyclonalantibody) and Table II (for monoclonal antibody) set forth below, plus30 minutes. The extra 30 minutes incubation period was added to equalizethis assay method with the other assay methods which required anadditional 30 minutes incubation time for a second added reagent.Following the incubation periods the agarose particles were washed byadditional of buffer and centrifuged. After removal of the washingliquid by aspiration, the resulting pellet of agarose particles was thencounted for bound ¹²⁵ I-labelled antibody. The counts obtained for eachof the complexes after the specified incubation time are set forth inTables I and II.

(2) Reverse Assay Method

Duplicate samples were run in which 100 μl of specimen (serum) was mixedwith 100 μl of ¹²⁵ I-labelled soluble antibody and incubated for thespecified times shown in Tables I and II. 100 μl a suspension ofantibody immobilized on agarose particles is then added and the mixturewas allowed to incubate for another 30 minutes. The agarose particleswere then washed and counted as in the simultaneous assay method. Thecounts are reported in Tables I and II.

(3) Forward Assay Method

Duplicate samples were run in which 100 μl of specimen (serum) was mixedwith 100 μl of a suspension of antibody immobilized on agarose particlesand incubated for the specified times shown in Tables I and II. Theagarose particles were then washed once by the addition of 2.5-3.0 ml ofbuffer which, after mixing, was centrifuged, and the liquid removed byaspiration. 100 μl of ¹²⁵ I-labelled soluble antibody was then added andthe mixture incubated an additional 30 minutes. The agarose particleswere then washed and counted as in the simultaneous assay method. Thecounts are reported in Tables I and II.

(4) Fast Forward Assay Method

The assay was performed, in duplicate, in a similar manner to theforward assay method except that the wash step between the initialincubation of specimen with antibody immobilized on agarose particlesand the addition of soluble ¹²⁵ I-labeled antibody was omitted.

The counts/minute for the duplicate controls and the duplicate assays ofthe samples containing IgE using polyclonal antibody and monoclonalantibody are shown in Tables I and II, respectively. These data wereused to prepare FIGS. I and II in the following way. The average of thecounts/minute for a control for a given incubation period was subtractedfrom the average of the counts for the corresponding IgE assay. Thedifference was calculated as a percentage of the total counts/minute oflabelled antibody added to the sample and is plotted on the Y axis asthe percentage of total counts/minute of antibody bound to the solidphase. The incubation time is plotted on the X axis.

                                      TABLE I                                     __________________________________________________________________________    Assay Results Using Polyclonal Antibody                                       Simultaneous                        Fast                                      Assay           Reverse Assay                                                                           Forward Assay                                                                           Forward Assay                             Incubation                                                                          Control                                                                            IgE  Control                                                                            IgE  Control                                                                            IgE  Control                                                                            IgE                                  Time (Hrs)                                                                          Samples                                                                            Samples                                                                            Samples                                                                            Samples                                                                            Samples                                                                            Samples                                                                            Samples                                                                            Samples                              __________________________________________________________________________    0.25  372,314                                                                            2705,2667                                                                          302,243                                                                            2568,2581                                                                          357,326                                                                            2092,2077                                                                          396,293                                                                            2271,2238                            0.50  348,265                                                                            2391,2366                                                                          284,262                                                                            2958,2999                                                                          288,233                                                                            1905,1817                                                                          ---,---                                                                            ----,----                            1.00  315,277                                                                            2793,2708                                                                          305,277                                                                            3154,3218                                                                          355,424                                                                            2157,2255                                                                          304,284                                                                            1789,1706                            2.00  342,356                                                                            2897,2887                                                                          290,274                                                                            3377,3212                                                                          302,314                                                                            1946,2019                                                                          288,312                                                                            1728,1867                            4.00  421,385                                                                            3696,3746                                                                           28,280                                                                            3413,3651                                                                          274,255                                                                            2019,2392                                                                          283,292                                                                            1720,1683                            6.00  447,436                                                                            4028,4101                                                                          296,281                                                                            3762,3643                                                                          241,267                                                                            1750,1452                                                                          301,257                                                                            1283,1424                            24.00 526,577                                                                            4564,4628                                                                          233,263                                                                            3651,3546                                                                          320,277                                                                            1553,1604                                                                          273,256                                                                            1450,1470                            __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________    Assay Results Using Monoclonal Antibody                                       Simultaneous                        Fast                                      Assay           Reverse Assay                                                                           Forward Assay                                                                           Forward Assay                             Incubation                                                                          Control                                                                            IgE  Control                                                                            IgE  Control                                                                            IgE  Control                                                                            IgE                                  Time (Hrs)                                                                          Samples                                                                            Samples                                                                            Samples                                                                            Samples                                                                            Samples                                                                            Samples                                                                            Samples                                                                            Samples                              __________________________________________________________________________    0.25  135,132                                                                            5610,5803                                                                          388,594                                                                            8407,8358                                                                          210,205                                                                            4618,4894                                                                          194,183                                                                            4859,4906                            0.50  558,459                                                                            7472,7115                                                                          240,231                                                                            8238,8271                                                                          223,228                                                                            4987,5273                                                                          198,197                                                                            5024,5152                            1.00  268,265                                                                            6289,6529                                                                          325,265                                                                            8010,8377                                                                          230,187                                                                            3454,4308                                                                          215,192                                                                            4887,4901                            2.00  282,275                                                                            6787,6784                                                                          255,302                                                                            7856,7644                                                                          226,197                                                                            4834,4268                                                                          192,210                                                                            4937,4944                            4.00  308,272                                                                            8150,8155                                                                          343,305                                                                            8017,7788                                                                          231,216                                                                            5269,4420                                                                          218,187                                                                            4929,5071                            8.00  549,667                                                                            8884,9494                                                                          698,850                                                                            7870,7358                                                                          226,361                                                                            2006,3631                                                                          405,243                                                                            3033,3713                            25.55 426,420                                                                            8669,9044                                                                          497,323                                                                            7037,7359                                                                          194,201                                                                            2465,2586                                                                          246,233                                                                            3945,2943                            __________________________________________________________________________

A comparison of the plots shown in FIG. 2 displaying the results ofassays using monoclonal antibody with those of FIG. 1 of assays usingusing polyclonal antibody shows that in each kind of assay,simultaneous, reverse, forward, and fast forward, the assay usingmonoclonal antibody was more sensitive as indicated by the higherpercentage of total counts bound to the solid phase with 100 IU IgE/mlspecimen. Unexpectedly, in the case of the simultaneous and reverseassays, we have found that those run with monoclonal antibody reachequilibrium more rapidly than does the corresponding assay usingpolyclonal antibody. Therefore, by using a monoclonal antibody in theseprocedures, the time for the assay can be reduced significantly beyondthe time saving achieved by merely eliminating a washing step. In thatregard, the reverse assay with monoclonal antibody reached equilibriumin less than one hour. The same assay run with polyclonal assay did notreach equilibrium until after 4 hours. Similarly, in the case ofsimultaneous assays, the assay using monoclonal antibody reachedequilibrium within 8 hours whereas the assay with polyclonal antibodydid not reach equilibrium within 24 hours. Accordingly, the presentinvention provides substantially more rapid and sensitive simultaneousand reverse assays than the prior art processes and eliminates theconcern that formation of a soluble "sandwich" complex will compete withformation of the desired insoluble complex.

In the foregoing discussion, the focus has been upon two site orsandwich assays in which one of the antibodies is insolubilized whilethe other is soluble in the medium analyzed. Other variations arepossible. A preferred variant employs antibodies bound to particles,such as particles of latex, in a manner which results in each particlecarrying a plurality of antibodies. When a quantity of particles towhich a first monoclonal antibody is bound is admixed with, for example,quantity of particles to which a second monoclonal antibody is bound, amilky suspension results. However, if a sample containing polyvalentantigen for which the antibodies are specific is introduced to thesuspension, agglomeration or agglutination of the particles will occurto form easily detectable particle clumps.

The visual detection of agglomerate formation can be used in a screeningtest for presence of the antigen. This detection can be aided bycoloring the particles carrying one monoclonal antibody differently fromthe particle carrying the other. However, the extent of agglomerationcan also be determined as a measure of the amount of antigen present inthe sample. For example, the change in turbidity can be measured usingstandard techniques such as nephelometry.

It is presently preferred to use latex particles to which the antibodyis covalently bound using techniques well known to those skilled in theart. However, other particulate supports can be used. Among these may bementioned silica, glass, cells, polyacrylamides, polymethyl methacrylateand agarose. Preferably, the particles vary in size between about 0.2μto about 10μ. Visual screening, however, requires particles of at leastabout 10μ.

In yet another variant, one of the antibodies is insolubilized on abead, test tube wall or other macroscopic solid support, and the otheris bound to small particles of latex or other suitable material. In thepresence of antigen, a sandwich of the antigen between themacroscopically bound antibody and the particle bound antibody willform. By, for example, coloring the particles, formation of the sandwichcan be determined visually. A fluorescent, enzymatic, radioactive orother label on the particle bound antibody can be used for quantitativedeterminations just as in the case of using a soluble antibody describedabove.

In another preferred variant of the two-site assay, at least one of twodifferent monoclonal antibodies is bound to an enzyme which catalyzes areaction involving a substance bound to the other monoclonal antibody toproduce either a detectable substance or in some other way interactswith the substance on the second antibody to permit detection of theantibody:antigen:antibody complex. Detection may be, for example, bycolorimetry, fluorimetry, luminescence, spectrophotometry, or the like.It will be appreciated that, using such techniques, neither antibodyneeds to be insolubilized, greatly simplifying the assay.

In a presently preferred embodiment, the substance on the secondantibody is also an enzyme and the assay employs the pair of enzymelabelled antibodies to catalyze sequential reactions, one of whichproduces a product required by the other. In those reactions, the twoantibodies are selected so that when they bind with the antigen, theyare sterically positioned so that the product of the first enzymaticreaction is generated in such close proximity to the second enzymelabelled antibody, that the second reaction occurs before significantdiffusion of the product of the first reaction into the surroundingmedium can take place.

This process can be illustrated using a pair of monoclonal antibodies,one of which is labelled with hexokinase (HK), the other withglucose-6-phosphate dehydrogenase (G-6-PDH), in the following series ofreactions. ##STR1##

The assay is conducted by adding to the sample containing the suspectantigen the labelled antibodies to the antigen, ATP, glucose and thecoenzyme NAD⁺. If the antigen is present, a complex as illustrated belowis formed: ##STR2##

The HK labelled antibody catalyzes the formation of glucose-6-phosphatein proximity to the G-6-PDH labelled antibody where it is converted togluconolactone-6-phosphate. The NADH formed in this reaction byreduction of NAD⁺ can be detected spectrophometrically because of thestrong absorption at 340 nm characteristic of a dihydronicotinamide.

The same conversion of glucose to gluconolactone-6-phosphate withformation of NADH also may occur in the medium itself catalyzed by theuncomplexed labelled antibodies, but at a much slower rate than when thetwo antibodies are positioned near each other in theantibody:antigen:antibody complex. Accordingly, an increase ofabsorption at 340 nm compared to a control sample confirms the presenceof antigen in the sample. The increase in absorption can also beco-related to the quantity of antigen in the complex.

Any other pair of suitable sequential enzymatically catalyzed reactionsmay be used in a two-site assay with appropriately labelled antibodiesto a suspect antigen. Among those may be mentioned the reaction ofglucose catalyzed with glucose oxidase to form glucono-δ-lactone andhydrogen peroxide followed by the reaction of the hydrogen peroxide witho-phenylenediamine catalyzed by peroxidase to produce a colored moiety.In this assay, one of the monoclonal antibodies is labelled with glucoseoxidase and the other with peroxidase. The intensity of the colorproduced compared to a control can be correlated to the presence and/oramount of antigen in the sample assayed. It will be appreciated thatother substances oxidizable to a colored moiety in the presence of anenzyme can be substituted for o-phenylenediamine.

Yet another suitable pair of sequential reactions using a pair ofantibodies to a desired antigen labelled, respectively, with NADoxidoreductase and luciferase is the following: ##STR3## RCHO istypically a straight chain aldehyde of 10 or more carbon atoms. Thegeneration of FMN*, an excited state of FMN, is followed by the emissionof a photon which can be detected photometrically for correlation with acontrol sample to indicate the presence and/or quantity of antigen in asample being assayed.

In another embodiment using a pair of antibodies labelled with enzymes,the product of the first enzymatically catalyzed reaction can be eitheran allosteric activator or inhibitor of the subsequent enzyme catalyzedreaction. An allosteric activator, rather than being consumed in thesecond reaction, interacts with the enzyme to increase its affinity fora substrate or to increase the rate of conversion of the substrate toproduct after the enzyme-substrate complex is formed. Allostericinhibitors, on the other hand, have the opposite effect and reduce theenzyme's affinity for a substrate or reduce the rate of conversion ofsubstrate to product. Allosteric inhibition may be of the competitive ornoncompetitive type.

An example of an assay involving allosteric activation employing a pairof antibodies labelled, respectively, with phosphofructokinase andphosphoenolpyruvate uses the following reaction scheme: ##STR4##

The fructose-1,6-diphosphate formed in reaction (1) allostericallyinteracts with the phosphoenolpyruvate carboxylase and activates itscatalysis of reaction (2), the formation of oxaloacetate from PEP.Reaction (3) occurs in the surrounding medium, i.e., there is nonecessity to bind the malate dehydrogenase to a third monoclonalantibody. The presence and/or quantity of suspect antigen is determinedby correlating the reduction in the absorption at 340 nm by NADH whichis oxidized in reaction (3) to NAD⁺.

An example of an assay involving allosteric inhibition employing a pairof antibodies labelled, respectfully, with aspartate amino transferase(AST) and phosphoenolpyruvate carboxylase can use the following reactionscheme: ##STR5##

The aspartate formed in reaction (1) inhibits the second reaction byallosterically interacting with the phosphoenolpyruvate carboxylase.This reduces the rate at which NADH is oxidized to NAD⁺. Therefore, thedecrease in the absorption at 340 nm exhibited by NADH can be correlatedto the presence and/or quantity of antigen in the sample being assayed,a smaller decrease than occurs with a control sample indicating thatantigen is present.

Those skilled in the art will appreciate that numerous other reactionpairs involving activation or inhibition of the second enzymaticallycatalyzed reaction can be substituted for the examples set forth abovefor use in a two-site assay. In another embodiment, only one of theantibody pairs is labelled with an enzyme, the second being labelledwith a substance, for example, that undergoes a reaction catalyzed bythe enzyme to produce a second product which can be detected and/orquantified by colorimetric, fluorimetric, luminescence,spectrophotometric or other technique. One such example uses a pair ofmonoclonal antibodies, one of which is labelled with peroxidase and theother with luminol, and takes advantage of the following reaction:##STR6##

The photon (hν) emitted by the reaction can be detected usingphotometric techniques and related to the presence and/or quantity of anantigen in a sample being assayed.

In yet another preferred variant of the two-site assay, the twomonoclonal antibodies are, respectively, conjugated with a fluorescingchromophore and a quenching chromophore which absorbs light at thewavelength emitted by the fluorescer. The two antibodies are selected sothat, when they combine with the antigen for which they are specific,the two chromophores are positioned close enough to permit the lightemitted by the fluorescer to be absorbed by the other chromophore.Usually, this will place them within about 100 angstroms of each otherand, preferably, within about 50 angstroms of each other. The selectionof suitable antibodies can be done through a screening procedure inwhich a mixture of fluorescent and quencher labelled monoclonalantibodies are contacted with a sample containing a known quantity ofantigen. Reduction of fluorescence is indicative that the twochromophores are positioned closely enough together.

Using fluorescence quenching, it is unnecessary to insolubilize eitherof the two antibodies. Quantitative measurements can be made simply bymeasuring the decrease in maximum fluorescence, i.e., the amount offluorescense exhibited by a control sample free of any antigen or bycomparing the fluorescence of the sample with that of control samplescontaining a known quantity of antigen. However, fluorescent-quenchingchromophore pairs can also be used in combination with the particleagglomeration technique and in the technique whereby one of theantibodies is insolubilized by being bound to a solid support such as atest tube wall or bead, since pairing of the fluorescent-quenchingchromophores will occur. A decrease in fluorescence again is indicativeof the presence or amount of antigen in the sample.

Suitable fluorescing and quenching chromophores and techniques forconjugating them with antibodies are described in U.S. Pat. No.4,174,384, the disclosure of which is incorporated by reference.Presently, it is preferred to use fluorescein and rhodamine as theflurorescer and quencher chromophores, respectively.

In the preceding discussion of our invention, we have describedtechniques of fluorescence quenching in which antibody pairs carryingthe necessary chromophores are caused to bond to an antigen, if presentin the sample being analyzed, in a steric arrangement which permits thequenching chromophore to absorb light emitted by the fluorescentchromophore. Quantitative determinations of the amount of antigenpresent are made by measuring the decrease in maximum fluorescence.

These techniques are well suited to determining the presence of antigenin a sample over a wide range of concentration. However, the smalldecreases in fluorescence which are associated with low antigenconcentration are hard to detect and measure accurately. By contrast,small increases in fluorescence are relatively easy to detect andmeasure accurately. Accordingly, in another aspect of our invention, weprefer to exploit the inhibition of quenching and measure increases influorescence.

To accomplish this in an assay for a particular antigen, quantities ofthe antigen and monoclonal antibody to the antigen are individuallylabelled with one or the other of the pair of fluorescent-quencherchromophores. The chromophore labelled antigen and antibody are thencombined to form a complex in which the fluorescent chromophore ispositioned so that the light it emits is absorbed by the quenchingchromophore. to achieve this the antigen may be labelled with thefluorescer and the antibody with the quencher or vice versa.

A sample suspected of containing the antigen being assayed is thencontacted with the chromophore labelled antigen and antibody. After asuitable incubation period, fluorescence is measured. If antigen ispresent in the assayed sample, it inhibits, at least in part, theformation of a complex between the chromophore labelled antigen and theantibody by itself forming a complex with the monoclonal antibody. Tothe extent this occurs, the fluorescer chromophore is no longerpositioned so that the light it emits is absorbed by the quenchingchromophore. This results in an increase in fluorescence. The increasein fluorescence can be measured and related to the concentration ofantigen in the sample undergoing analysis by comparison with thefluorescence exhibited by control samples free of antigen or containingknown amounts of antigen.

From the foregoing, it will be apparent that the chromophore labelledantigen:antibody complex may be a soluble one. However, it is presentlypreferred to employ chromophore labelled antigen and monoclonal antibodywhich are bound to latex or other suitable particles, for example, thoselisted above, of a size that will form agglomerates when the complex isformed. Particles varying in size from about 0.2 to about 10μ areusually suitable for this purpose. When an unknown sample containing thesuspect antigen is contacted with the agglomerate forming particles ofantibody and antigen, inhibition of agglomeration will occur because ofsample antigen combining with particle-bound antibody. An increase influorescence will result, since quenching can no longer occur, which canbe detected and measured to correlate with the amount of antigen in thesample by comparison with the fluoresence observed for a samplecontaining a known quantity of antigen.

It is also within the scope of our invention to employ bound antigen andparticle bound monoclonal antibody in an assay that directly measuresthe inhibition of agglomeration. In this technique, neither the antigennor antibody is labelled. When a sample containing antigen is contactedwith the particles, inhibition of agglomeration during the incubationperiod will occur. This results in, at least, a partial reduction in theagglomerate formation. The inhibition is detected using nephelometry orother techniques for measuring turbidity. The decrease in turbidity canbe correlated to the amount of antigen in the sample.

The foregoing description of the invention and the examplesdemonstrating the application of the invention to assays for IgE are butexemplary of the various ways the invention can be utilized. That othervariations will be useful will be apparent to those skilled in the art.Therefore, the present invention is to be considered limited only by theappended claims.

We claim:
 1. In an immunometric assay process to determine the presenceor concentration of an antigenic substance in a fluid sample comprisingforming a ternary complex of the antigenic substance, a first antibodyand a second antibody bound to the antigen at a difference site than thefirst antibody by contacting the sample with said first and secondantibodies, the improvement comprising employing a measured amount of amonoclonal antibody having an affinity for the antigenic substance of atleast about 10⁸ liters/mole for each of said first and secondantibodies.
 2. A process according to claim 1 wherein a fluorescentchromophore is bonded to said first antibody and a chromophore capableof absorbing light at the wavelength emitted by the fluorescentchromophore is bonded to said second antibody, the monoclonal antibodiesbeing capable of binding to the antigenic substance at sites whichpermit the light emitted by the fluorescing chromophore to be absorbedby the quenching chromophore.
 3. A process according to claim 2 whereinthe sample is contacted with a solution containing the first and secondantibodies to form the ternary complex and the intensity of fluorescencedetermined and compared to the fluorescence of a standard sample free ofsaid antigen or containing said antigen in a known amount.
 4. A processaccording to claim 2 wherein the first antibody is bound to a solidcarrier that is insoluble in the fluid sample and said second antibodyis soluble in the fluid sample.
 5. A process according to claim 4wherein the fluid sample is simultaneously contacted with said first andsecond antibodies to form an insoluble ternary complex and the intensityof fluorescence of the ternary complex determined and compared to thefluorescence of a standard sample free of said antigen or containingsaid antigen in a known amount.
 6. A process according to claim 2wherein the second antibody is bound to a solid carrier that isinsoluble in the fluid sample and said first antibody is soluble in thefluid sample.
 7. A process according to claim 6 wherein the fluid sampleis simultaneously contacted with said first and second antibodies toform an insoluble complex and the intensity of fluorescence of the fluiddetermined and compared to the fluorescence of a standard sample free ofsaid antigen or containing said antigen in a known amount.
 8. A processaccording to claims 4 or 6 wherein the sample is first contacted withthe first antibody to form an antibody:antigen binary complex and thencontacted with the second antibody to form the ternary complex and theintensity of the fluorescence of the complex or the fluid determined andcompared to a standard sample free of said antigen or containing theantigen in a known amount.
 9. A process according to claims 2, 3, 4, 5,6, or 7 wherein the fluorescent chromophore is fluorescein and thechromophore capable of absorbing emitted light is rhodamine.
 10. Aprocess according to claim 8 wherein the fluorescent chromophore isfluorescein and the chromophore capable ot absorbing emitted light isrhodamine.
 11. A process according to claim 1 wherein said firstantibody is bound to particles insoluble in the fluid sample and saidsecond antibody is bound to particles insoluble in the fluid sample andwherein the sample is contacted with a suspension of the particles for atime sufficient to cause formation of the ternary complex wherebyagglomeration of the particles bound to said first and second antibodiesoccurs.
 12. A process according to claim 11 wherein the size of theparticles is in the range of from about 0.2μ to about 10μ.
 13. A processaccording to claim 12 wherein the particles are selected from the groupconsisting of particles of latex, silica, glass, cells, polyacrylamide,polymethyl methacrylate and agarose.
 14. A process according to claim 13wherein the particles are latex particles.
 15. A process according toclaims 11, 12, 13, or 14 wherein the particles binding the firstantibody are of different color than the particles binding the secondantibody.
 16. A process according to claims 12, 13, or 14 wherein thesize of the particles is in the range of from about 1.0 to 10μ.
 17. Aprocess according to claim 15 wherein the size of the particles is inthe range of from about 1.0 to 10μ.
 18. A process according to claims11, 12, 13, or 14 wherein the turbidity of the sample after formation ofthe ternary complex is determined and related to the turbidity of acontrol sample known to be free of the antigen or to contain a knownamount of the antigen.
 19. A process according to claims 11, 12, 13, or14 wherein a fluorescent chromophore is bound to said first antibody anda chromophore capable of absorbing light at the wavelength emitted bythe fluorescent chromopore is bound to the second antibody and wherein,after said contacting, the intensity of fluorescence is determined andcompared to the fluorescence of a standard sample free of said antigenor containing said antigen in a known amount.
 20. A process according toclaim 19 wherein the fluorescent chromophore is flurescein and thechromophore capable of absorbing emitted light is rhodamine.
 21. Aprocess according to claim 1 wherein an enzyme is bound to the firstantibody and a substance is bound to the second antibody whereby theenzyme interacts with the substance to permit detection of theantibody:antigen:antibody complex.
 22. A process according to claim 21wherein the substance bound to the second antibody is an enzyme.
 23. Aprocess according to claim 22 whereby the first antibody bound enzymecatalyzes a reaction which produces a product required by a reactioncatalyzed by the second antibody bound enzyme.
 24. A process accordingto claim 23 whereby the product of the reaction catalyzed by the firstbound enzyme is consumed in the reaction catalyzed by the second boundenzyme.
 25. A process according to claim 23 whereby the product of thereaction catalyzed by the first bound enzyme allosterically interactswith the second bound enzyme.
 26. A process according to claim 25whereby the product of the reaction catalyzed by the first bound enzymeallosterically activates the second bound enzyme.
 27. A processaccording to claim 25 whereby the product of the reaction catalyzed bythe first bound enzyme allosterically inhibits the second bound enzyme.28. A process according to claims 23, 24, 25, 26 or 27 wherein thereaction catalyzed by the second antibody bound enzyme produces adetectable product.
 29. A process according to claim 28 wherein theformation of the detectable product is detected by colorimetry,fluorimetry, luminescence or spectrophotometry.
 30. A process accordingto claims 23, 24, 25, 26 or 27 wherein the reaction catalyzed by thesecond antibody bound enzyme consumes a detectable substance.
 31. Aprocess according to claim 30 wherein the consumption of the detectablesubstance is detected by colorimetry, fluorimetry, luminescence orspectrophotometry.
 32. A process according to claim 21 wherein thesubstance on the second antibody undergoes a reaction catalyzed by thefirst antibody bound enzyme.
 33. A process according to claim 32 whereinthe reaction produces a substance detectable by colorimetry,fluorimetry, luminescence or spectrophotometry.